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Tetrapyrrole Synthesis of Photosynthetic Chromerids
Is Likely Homologous to the Unusual Pathway of
Apicomplexan Parasites
C W
Luděk Kořený,a Roman Sobotka,b Jan Janouškovec,c Patrick J. Keeling,c and Miroslav Obornı́ka,b,1
a Institute
of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, and Faculty of Science, University of
South Bohemia, Branišovská 31, 37005 České Budějovice, Czech Republic
b Institute of Microbiology, Academy of Sciences of the Czech Republic, 37981 Třeboň, Czech Republic
c Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
Most photosynthetic eukaryotes synthesize both heme and chlorophyll via a common tetrapyrrole biosynthetic pathway
starting from glutamate. This pathway was derived mainly from cyanobacterial predecessor of the plastid and differs from
the heme synthesis of the plastid-lacking eukaryotes. Here, we show that the coral-associated alveolate Chromera velia, the
closest known photosynthetic relative to Apicomplexa, possesses a tetrapyrrole pathway that is homologous to the unusual
pathway of apicomplexan parasites. We also demonstrate that, unlike other eukaryotic phototrophs, Chromera synthesizes
chlorophyll from glycine and succinyl-CoA rather than glutamate. Our data shed light on the evolution of the heme biosynthesis in parasitic Apicomplexa and photosynthesis-related biochemical processes in their ancestors.
INTRODUCTION
Apicomplexa is a diverse group of parasites, which infect various
animals, including humans. Plasmodium, the causative agent of
malaria kills millions of people every year (Alemu et al., 2011).
Most of the Apicomplexa have been shown to contain a secondary four membrane–bound nonphotosynthetic plastid (apicoplast) that is essential for the parasite survival (reviewed in
Obornı́k et al., 2009 or Kalanon and McFadden, 2010). The
presence of this organelle suggested a photosynthetic ancestry
for apicomplexans almost two decades ago; however, no close
photosynthetic relative was identified until very recently (Moore
et al., 2008). This novel alga, named Chromera velia, contains
ultrastructural features typical for apicomplexans and related
alveolates, such as cortical alveoli, underlying microtubular
corset, and an apical microtubular cone (Moore et al., 2008;
Obornı́k et al., 2011), but also possesses a single four membrane–bound plastid. Because Chromera and apicomplexans
are sisters in both nuclear and plastid gene phylogenies and also
share several unique plastid-related features (together with
another alveolate known as CCMP3155), their extant plastids
clearly share a common evolutionary origin (Janouškovec et al.,
2010). Discovery of C. velia therefore gives an unprecedented
opportunity to uncover the course of early apicomplexan evolu-
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Miroslav Obornı́k
([email protected]).
C
Some figures in this article are displayed in color online but in black
and white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.111.089102
tion and improve our understanding of how a free-living alga
transformed into an obligate parasite. Of particular interest is the
transformation of the plastid organelle and metabolic pathways
commonly found in photosynthetic eukaryotes that have been
retained in the apicoplast. Three such pathways, mediating the
synthesis of fatty acids, isoprenoids, and tetrapyrroles, have
been shown to be indispensable for the apicomplexan parasites
survival and are considered potential drug targets in treating
malaria and other diseases (Ralph et al., 2004).
Tetrapyrrole biosynthesis ranks among the most fundamental
pathways in living systems. It serves the synthesis of heme, a
molecule central to the oxidative and energy metabolism, and
chlorophyll formation in photoautotrophs. Synthesis of tetrapyrroles is conserved generally among all three domains of life, with
the exception of the first step, the synthesis of d-aminolevulinic
acid (ALA) (Panek and O’Brian, 2002). Primarily heterotrophic
eukaryotes, such as animals and fungi, synthesize ALA in the
mitochondrion through the condensation of Gly and succinylCoA in a C4 pathway catalyzed by ALA-synthase (ALAS). This
enzyme is otherwise found only in a-proteobacteria, which
supports its mitochondrial origin (Figure 1A). ALA is exported to
the cytosol, where the next four to five biosynthetic steps are catalyzed by enzymes originating from the ancient eukaryotic nucleus, with the possible exception of uroporphyrinogen synthase
(UROS) (Figures 1B and 2; see Supplemental Figure 1 online).
The final steps take place back in the mitochondrion, where the
majority of heme is needed for respiratory cytochromes (Dailey
et al., 2005) (for details, see summary in Figure 3).
Photosynthetic eukaryotes synthesize ALA from Glu bound to
tRNAGlu through the C5 pathway consisting of two steps catalyzed by glutamyl-tRNA reductase (GTR) and glutamate 1-semialdehyde aminotransferase (GSA-AT). The remaining steps are
catalyzed by the same enzymes as in heterotrophs, but the entire
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Figure 1. Phylogenetic Trees of Ferrochelatase and ALA-Synthase.
Tetrapyrrole Synthesis in Chromera velia
pathway is localized in the plastid, and most of the involved
enzymes originate from its cyanobacterial ancestor (Obornı́k and
Green, 2005) (Figure 3B). This compartmentalization reflects the
major need for the tetrapyrroles in the plastid, where chlorophyll
and most of the synthesized heme are required for photosynthetic functions (Papenbrock et al., 1999; Castelfranco and
Jones, 1975).
Heme synthesis in Apicomplexa follows a unique route distributed among three cellular compartments. ALA produced in
the mitochondrion by the C4 pathway is exported to the apicoplast, where the next three to four steps take place. The sixth
enzymatic reaction is situated in the cytosol, while the final two
steps are catalyzed in the mitochondrion (Nagaraj et al., 2010a)
(Figure 3B). The apicomplexan pathway is an evolutionary mosaic combining pathways from both heterotrophic and photosynthetic eukaryotes. ALAS, uroporphyrinogen decarboxylase
(UROD), and coproporphyrinogen oxidase (CPOX) were present
in the host cell prior to the endosymbiotic origin of the apicoplast.
The remaining enzymes (apart from ferrochelatase) are related to
homologs from photosynthetic eukaryotes, suggesting their
origin in algal endosymbiont (Figure 3B). Ferrochelatase (FeCH)
clusters with proteobacteria and probably comes from a nonendosymbiotic horizontal gene transfer (Sato and Wilson, 2003)
(Figure 1B).
To shed more light on the evolution of the unusual tetrapyrrole
synthesis in apicomplexan parasites, we investigated this pathway in C. velia, a closely related photosynthetic alga.
RESULTS AND DISCUSSION
Identification and Phylogeny of Tetrapyrrole Biosynthesis
Genes in C. velia
Fragments of all tetrapyrrole biosynthesis enzymes, with the
single exception of protoporphyrinogen oxidase (PPOX), were
identified within the sequences of an ongoing 454 genome
survey of C. velia (Janouškovec et al., 2010), and full-length
transcripts were obtained by rapid amplification of cDNA ends
(RACE). Interestingly, a gene encoding ALAS, the enzyme of the
C4 pathway, was found (Figure 1A), but we failed to identify GTR
or GSA-AT, the C5 pathway enzymes used by other photoautotrophs (Figure 3B).
Phylogenetic analyses show that the tetrapyrrole biosynthetic
pathway of C. velia is a mosaic composed of genes of different
origins. Although all the proteins are encoded in the nucleus of C.
velia, only few of them display a phylogenetic pattern corresponding to the phylogeny of the host cell. The others seem to
originate from distinct genomes (e.g., the plastid) and were
horizontally transferred to the host nucleus. C. velia possesses
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genes that have the same origins as the genes of heme biosynthesis from Apicomplexa, indicating that these pathways share
the same evolutionary history (Figures 1 to 3; see Supplemental
Figure 1 online). However, in contrast with the pathway of
apicomplexans where each step is catalyzed by a single enzyme,
Chromera encodes four additional proteins (two URODs, CPOX,
and FeCH), sharing an evolutionary history with other photosynthetic eukaryotes (Figure 3B). ALAS clusters with homologs from
heterotrophic eukaryotes and a-proteobacteria, implying its
origin in the mitochondrion (Figure 1A). ALAD, on the other
hand, originates from the plastid as it groups with photosynthetic
eukaryotes and cyanobacteria (see Supplemental Figure 1A
online), which is also true for one UROD and one FeCH (Figures
1B and 2). PBGD of C. velia branches together with photosynthetic eukaryotes, suggesting its origin in the endosymbiont.
However, this clade is not related to cyanobacteria but is nested
within a-proteobacteria, which indicates its origin in the mitochondrion of the primary host (see Supplemental Figure 1B
online). Similarly, UROS and one CPOX cluster with photosynthetic eukaryotes but not with cyanobacteria, and the primary
origins of these particular enzymes are not clear (see Supplemental Figures 1C and 1D online). Apart from the cyanobacterial
UROD, there are two other URODs encoded in the genome of C.
velia, which are of eukaryotic origin (Figure 2). One comes from
the nucleus of the primary alga as it groups with both primary and
secondary algae, and the other one represents the original
enzyme of the final host, which is also the case of one CPOX
(see Supplemental Figure 1D online). The second FeCH is closely
related to the same enzymes from Apicomplexa and was likely
horizontally transferred from some proteobacteria to the ancestor of Chromera and Apicomplexa (Sato and Wilson, 2003; Figure
1B). Regardless of the primary origins of the enzymes, it is clear
that ALAS, one UROD, one CPOX, and one FeCH represent the
original heme pathway of the heterotrophic host, but ALAD,
PBGD, UROS, two URODs, one CPOX, and one FeCH are
derived from the endosymbiotic alga that was transformed into
the plastid (see Figure 3B for the summary of gene origins).
Predicted Subcellular Location of C. velia Tetrapyrrole
Biosynthesis Proteins
The analysis of 59 ends of transcript sequences of all enzymes in
question revealed that, unlike the others, ALAS possesses an
N-terminal extension that resembles the mitochondrial transit
peptide. This presequence is similar to those of other eukaryotic
ALASs (see Supplemental Figure 2 online), which were experimentally shown to be present in mitochondria in various eukaryotes from different lineages including Apicomplexa (Volland and
Felix, 1984; Dailey et al., 2005; Sato et al., 2004; Wu, 2006); thus,
we believe the C. velia ALAS is most likely also located in the
Figure 1. (continued).
Bayesian phylogenetic trees as inferred from ALAS (A) and ferrochelatase (B) amino acid sequences, and the enzymes of the first and last step of the
heme synthesis. Numbers above branches indicate Bayesian posterior probabilities. The trees demonstrate mitochondrial origin of ALAS (A) and
uncovered enigmatic origins of the apicomplexan ferrochelatase (B).
[See online article for color version of this figure.]
Figure 2. Bayesian Phylogenetic Tree as Inferred from UROD Amino Acid Sequences.
The tree well demonstrates the complex symbiotic origin of C. velia. Particular UROD enzymes appear to originate from cyanobacteria, the primary host
nucleus (nucleus of a eukaryotic alga involved in secondary endosymbiosis), and secondary host nucleus (nucleus of a heterotrophic eukaryote that
engulfed the alga). Numbers above branches indicate Bayesian posterior probabilities.
[See online article for color version of this figure.]
Tetrapyrrole Synthesis in Chromera velia
mitochondrion. Such localization corresponds well to the origin
of ALAS in the a-proteobacterial predecessor of the mitochondrion (Schulze et al., 2006). This origin is shared by all eukaryotes,
including C. velia (Figure 1A). Furthermore, two heme regulatory
motifs in the N-terminal presequence of C. velia ALAS (see
Supplemental Figure 3 online) suggest the same regulation of the
tetrapyrrole synthesis as in heterotrophs, which is the hememediated inhibition of ALAS import into mitochondrion (Zhang
and Guarente, 1995; Dailey et al., 2005). In addition to that, ALAS
directly uses succinyl-CoA, the product of mitochondrially located citric acid cycle, as a substrate; thus, its localization to a
different compartment would be rather hard to imagine. This
corresponds to the fact that ALAS has never been localized
elsewhere than to mitochondrion.
Except for ALAS, all the other enzymes of heme biosynthesis in
C. velia possess typical N-terminal bipartite leader sequences
that consist of a signal peptide (SP) and a transit peptide (TP) and
are required for targeting to the complex plastids (Kroth, 2002).
These leaders are highly similar to those found in heterokont
algae and Apicomplexa; thus, all of these proteins seem to be
targeted to the plastid of C. velia. SPs were easily recognized by
in silico predictions with extremely high probabilities (from 0.966
to 1.000) in each of the proteins, using both approaches available
to date, the neural networks (data not shown), and the hidden
Markov models (Table 1). After removal of SPs, all the proteins
still possess significantly long N-terminal presequences (compared with their bacterial counterparts), which display chemical
properties typical for TPs (von Heijne et al., 1989), and most of
them (eight out of 10) were recognized as TPs by the prediction
tool (Table 1). Predictions of TPs are complicated by the fact that
mitochondrial (mTP) and plastidial transit peptides (cTPs) tend to
be similar in algae with complex plastids. However, it is well
established that in secondary algae it is exclusively the combination of SPs and TPs that decides the plastid localization of a
protein, whereas the mitochondrially located proteins are recognized by the presence of TP alone. This leads to a conclusion
that there is no selection pressure to distinguish cTP from mTP in
secondary algae (Jiroutová et al., 2007; Rao et al., 2008). This
means that the SP can be followed by virtually any presequence
that has the general chemical properties of a TP to target the
proteins to the complex plastids.
It has been suggested that it is the number of membranes
surrounding the plastids that decides the mechanism of protein
targeting (Nassoury et al., 2003). In contrast with double membrane plastids of primary algae and plants, the complex plastids
of secondary algae are surrounded by additional membranes,
and the proteins are therefore imported into this organelle by a
distinct route: SP targets the proteins to endoplasmic reticulum
and consequently across the outermost plastid membrane that is
topologically identical with the endoplasmic reticulum. When the
SP is cleaved off, the TP is recognized by the plastid import
machinery and transfers the proteins through the two innermost
membranes of the complex plastid, which correspond to the
primary plastid envelope (Lang et al., 1998; Waller et al., 1998;
McFadden, 1999; Foth et al., 2003). Although the particulars of
the import mechanism slightly differ among taxa, the use of
bipartite leader sequence seems to be a universal mark that
guides proteins into the complex plastids of all secondary algae.
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Such protein import has been already experimentally proven
in apicomplexans (Waller et al., 1998), diatoms (Lang et al.,
1998), euglenozoans (Shashidhara et al., 1992), dinoflagellates
(Nassoury et al., 2003), cryptophytes (Gould et al., 2006), and
chlorarachniophytes (Hirakawa et al., 2009). Since phylogenies
based on both nuclear and plastid genes congruently place C.
velia within chromalveolates close to apicomplexan parasites
(Janouškovec et al., 2010), it is bordering on certainty that the
same targeting mechanism operates in C. velia as well.
Our predicted localizations of these enzymes into the plastid of
C. velia in most cases reflect their origin in the algal symbiont.
Most of them are most closely related to the enzymes of other
photosynthetic eukaryotes, where the entire pathway is located
to the plastid (Obornı́k and Green, 2005). The proteobacteria-like
FeCH of C. velia that is not present in the genomes of other
phototrophs but is shared with apicomplexan parasites seems
to be located in the plastid of C. velia as well. This is rather
surprising since this enzyme was shown to localize to the
mitochondrion of Apicomplexa. However, in C. velia, it possesses
clear bipartite N-terminal presequence (;90 amino acids long),
whereas its homolog in Plasmodium falciparum bears much
shorter presequence (;20 amino acids), which represents the
mTP alone. Similarly, the cytosolic CPOX of P. falciparum lacks
any N-terminal presequence when compared with bacterial homologs, while the N terminus of the CPOX of the same origin
(secondary host nucleus) in C. velia is 65 amino acids longer, and
both the SP and TP were predicted in this presequence (Table 1).
These marked differences in the N-terminal sequences of the
closely related enzymes between C. velia and P. falciparum are
very likely the result of a selection pressure for different localizations. The presence of two distinct ferrochelatases in the plastid is
not unprecedented; it was experimentally demonstrated in plants
(Woodson et al., 2011).
Although the presence of a bipartite targeting presequence is
generally considered as an evidence of localization to complex
plastids, dual targeting to other compartment cannot be ruled out in
some cases. However, testing this possibility would require direct
experimental localization of these enzymes, which is not available
at the moment due to the lack of transfection system for C. velia.
The multiple lines of evidence involving presence of putative
targeting sequences, molecular phylogeny of the studied enzymes, as well as arrangement of the pathway in other phototrophic
eukaryotes examined so far lead us to the prediction of intracellular
location of enzymes involved in tetrapyrrole biosynthesis in C. velia,
as shown in Figure 3B.
C. velia Synthesizes Tetrapyrroles from Gly
and Succinyl-CoA
Our analysis of genomic information leads to the prediction that
C. velia is unique in being able to synthesize chlorophyll from Gly
and succinyl-CoA via the C4 pathway. Other photoautotrophs
synthesize chlorophylls using the C5 pathway. To test this
prediction, we supplied C. velia with either 14C-Gly or 14C-Glu
and monitored the incorporation of labeled amino acids into
the chlorophyll. The cyanobacterium Synechocystis 6803, with
a well-characterized C5 pathway, was used as a control. As
expected, C. velia uses Gly as a substrate for chlorophyll
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synthesis in contrast with Synechocystis, which only incorporates
Glu into chlorophyll (Figure 4). We also found that C. velia metabolizes U-14C-Glu into chlorophyll, which could be due to the parallel
use of the C5 pathway but could also be due to the easy conversion
of this amino acid into succinyl-CoA in the mitochondrion. To distinguish between these two possibilities, C. velia was grown in the
presence of 1-14C-Glu labeled specifically on the carboxyl group,
which is metabolized to eight carbons of chlorophyll through C5
pathway but is released as CO2 when Glu enters the citric acid
cycle. In this case, we detected only traces of labeled chlorophyll
when compared with 2-14C-Gly used as a control (Figure 4).
Implications of the Evolution of Tetrapyrrole Synthesis
Figure 3. Tetrapyrrole Biosynthetic Pathways of Various Eukaryotes.
Distribution of different types of the tetrapyrrole biosynthetic pathway (B)
among chromalveolates (A). (1) Canonical C4 pathway of heterotrophic
eukaryotes; localization differs between animal cells (Dailey et al., 2005)
and yeast (Camadro et al. 1986). (2) Canonical C5 pathway that is shared
by most of the photosynthetic eukaryotes. (3) Tetrapyrrole pathway of
C. velia with putative localizations based on in silico predictions using
SignalP and TargetP, respectively (Emanuelsson et al., 2007). (4) Heme
biosynthesis of Apicomplexa; localization differs between P. falciparum
and Toxoplasma gondii (Sato et al., 2004; Nagaraj et al., 2009a, 2009b,
2010a, 2010b; van Dooren et al., 2006; Wu, 2006; Shanmugam et al.,
2010). Numbers of the individual steps of the synthesis represent the
enzymes: 1, ALAS; 1a, GTR; 1b, GSA-AT; 2, ALAD; 3, PBGD; 4, UROS; 5,
UROD; 6, CPOX; 7, PPOX; 8, FeCH. The abbreviations of the enzyme
names are explained in the text. The colors of the enzymatic steps stand
for the origin of the genes, which are inferred from the phylogenetic trees
(see Supplemental Figure 1 online). *Only incomplete information is
available: Schizochytrium possesses ALAS, Perkinsus uses the C4
pathway as well as ALAD and UROD of the same origin as in primary
heterotrophs. From Oxyrrhis marina, the sequence of the gene encoding
PBGD shows a relation to the phototrophic PBGDs.
Tetrapyrrole biosynthesis in C. velia represents an evolutionary
intermediate between the homologous pathways in other photosynthetic eukaryotes and parasitic apicomplexans. Targeting
presequences of C. velia enzymes suggest that the bulk of the
tetrapyrrole biosynthesis takes place in the plastid, as is the case in
other photosynthetic eukaryotes. This localization is in agreement
with the primary need for tetrapyrrole products in plastidic chlorophyll and heme biosynthesis, which most likely represents the
state of the photosynthetic ancestor of apicomplexans. Moreover,
C. velia contains multiple enzyme isoforms in at least three steps of
the pathway, and evolutionary histories of these proteins suggest
they were also present in the ancestor of apicomplexans. When
photosynthesis was lost in the ancestor of apicomplexans, all
enzyme isoforms were reduced to a single copy, and the last steps
of the pathway catalyzed in the plastid were relocated to the
cytosol (CPOX) and the mitochondrion (PPOX and FeCH).
This recompartmentalization is especially interesting because
it readily illustrates how different cellular demands resulting from
a life mode change can dramatically reshape organelle metabolism. In photosynthetic eukaryotes, chlorophyll is synthesized at
a much higher rate than heme, and most of the heme is used for
photosynthetic functions in the plastid. It is used in the cytochromes
of photosystems and also serves as a precursor for the synthesis of
billin pigments (Papenbrock et al., 1999; Castelfranco and Jones,
1975), while the mitochondrial heme requirements comprises only a
fraction of the bulk tetrapyrrole product needed in the chloroplast. In
heterotrophic eukaryotes and parasites, however, the heme biosynthesis in mitochondria is the principal tetrapyrrole sink. Tetrapyrrole biosynthesis in apicomplexans therefore provides a nice
example how a metabolic pathway and trophic mode directly link
through compartmentalization of the last biosynthetic steps, which
generate products where most needed.
Interestingly, this pathway also gives us a unique opportunity
to understand the recompartmentalization process, which is
intimately linked to acquisition of a second ferrochelatase isoform from proteobacteria by the ancestor of apicomplexans and
C. velia. The shared presence of this enzyme in C. velia and
apicomplexans shows that its uptake was not an adaptive
response to switch to parasitism in apicomplexans but a more
ancestral condition that may have facilitated such a lifestyle
change. More specifically, it is likely that presence of two
functionally overlapping ferrochelatases lead to mitochondrial
retargeting of the proteobacterial form in the apicomplexan
ancestor without harmfully affecting tetrapyrrole biosynthesis in
Tetrapyrrole Synthesis in Chromera velia
Table 1. In Silico Predictions for the Presence of SPs and TPs in the
Protein Sequences of C. velia
Protein/GenBank
Accession No.
SP (SignalP)
TP (TargetP)
ALAS/HQ222925
ALAD/HQ222926
PBGD/HQ222927
UROS/HQ222928
URODa/HQ222929
URODb/HQ222930
URODc/HQ222931
CPOXa/HQ222932
CPOXb/HQ222933
FeCHa/HQ222934
FeCHb/HQ222935
Not Predicted
0.994 (15 aa)
0.996 (18 aa)
0.985 (17 aa)
1.000 (25 aa)
1.000 (22 aa)
0.992 (21 aa)
0.966 (25 aa)
1.000 (23 aa)
0.991 (32 aa)
0.971 (31 aa)
Not Predicted (112 aa)a
0.689 (cTP; 67 aa)
0.638 (mTP; 22 aa)
0.771 (mTP; 16 aa)
not predicted (43 aa)b
0.929 (cTP; 42 aa)
0.587 (mTP; 28 aa)
0.658 (mTP; 27 aa)
0.924 (cTP; 28 aa)
0.806 (mTP; 45 aa)
not predicted (60 aa)b
aa, amino acids.
aThe mitochondrial TP of ALAS is not predictable by TargetP in C. velia
as well as in other eukaryotes, including apicomplexans, in which the
mitochondrial location has been experimentally proven; however, the
protein possesses a long N-terminal extension, when compared to
bacterial sequences, which resembles the mitochondrial TPs of other
eukaryotic ALAS enzymes.
bAlthough the TPs were not predicted for some of the proteins that have
SPs, they possess significant N-terminal extensions (after removal of
SP) compared to bacterial sequences. Their approximate length is
noted in parentheses.
the plastid. After the acquisition of parasitism, the need for
plastid tetrapyrroles became redundant and the canonical plastidic ferrochelatase was lost, while the proteobacterial ferrochelatase took over the tetrapyrrole supply for mitochondrial heme.
In conclusion, this process illustrates how functional enzyme
duplication mediated by horizontal gene transfer can facilitate
relocation of a biosynthetic step between organelles.
To gain broader understanding of the evolution of the unusual
pathways in Apicomplexa and C. velia, we examined the tetrapyrrole pathways of other chromalveolates. The whole-genome
searches and our phylogenetic data revealed that heterotrophic
chromalveolates in which plastids have not been found, such as
ciliates and oomycetes, use the C4 pathway to synthesize ALA,
and all the genes involved in the tetrapyrrole synthesis have the
same origins as in primary heterotrophs. By contrast, the photosynthetic stramenopiles, cryptophytes, and haptophytes synthesize tetrapyrroles using the C5 pathway, as do primary algae
(Figure 3). The same is probably true for dinoflagellates, as only
the C5 pathway genes were found in the available expressed
sequence tags of eight different species of this taxon. Since
plastids of alveolates and heterokont algae are believed to share
a common ancestry (Janouškovec et al., 2010), the presence of a
heterotrophic-like pathway in some chromalveolates (Figure 3A)
can be explained by the early loss of the plastid and the
coexistence of separate tetrapyrrole pathways in these lineages
before this event. In the lineage represented by C. velia and
apicomplexan parasites, both pathways were combined to yield
their unique pathways (Figure 3). Both tetrapyrrole pathways that
are suggested to operate in the ancestral lineages of Chromalveolates are still found in photosynthetic euglenids, which arose
through an independent secondary symbiosis involving a green
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alga. Euglena gracilis still possesses the original C4 pathway of the
host cell that produces heme in the mitochondrion, while the
plastid-localized C5 pathway that was derived from the algal
endosymbiont serves only for the synthesis of chlorophyll and
plastidial heme (Weinstein and Beale, 1983; Kořený and Obornı́k,
2011). Consequently, Euglena seems to be the only known eukaryotic phototroph that can lose its plastid and live as a heterotroph without external source of heme (Osafune and Schiff, 1983).
As mentioned elsewhere (Obornı́k et al., 2009), the plastid could be
lost only during the early stages of secondary endosymbiosis
before the host cell delegated essential functions to this organelle.
However, when the plastid took over the tetrapyrrole synthesis
and the original pathway disappeared (as happened in most of
photosynthetic eukaryotes), the plastid could no longer be lost
without first acquiring some other source of tetrapyrroles. This
corresponds well to the findings that the only apicomplexans
without detectable plastids are those that do not synthesize their
own heme (Mather and Vaidya, 2008; Mogi and Kita, 2010;
Templeton et al., 2010).
METHODS
Sequence Retrieval and Phylogenetic Analyses
Partial sequences of the genes of the heme biosynthesis pathway were
searched in the sequence data of the 454 genome sequencing surveys of
Chromera velia and CCMP3155 (Janouškovec et al., 2010) by BLAST
2.2.18 (http://blast.ncbi.nlm.nih.gov). Complete coding sequences were
amplified from the cDNA of C. velia by 39 RACE and 59 RACE using the
FirstChoice RLM-RACE kit (Ambion), cloned, and sequenced. For each
enzyme, appropriate homologs were identified by BLAST and downloaded from Web sources (see Supplemental Table 1 online), aligned
using MAFFT (Katoh and Toh, 2008), manually edited using BioEdit (Hall,
1999), and used for further phylogenetic analyses (see Supplemental
Data Sets 1 to 3 online). Bayesian trees were computed using PhyloBayes
3.2d under CAT-GTR evolutionary model (Lartillot and Philippe, 2004). For
each analysis, two independent chains were run for a sufficient number of
steps after the chains converged. The convergence of two independent
chains was assessed based on bpcomp analysis (PhyloBayes 3.2d). The
burnin value was chosen according to the same analysis (usually 20% of
Figure 4. Incorporation of Radiolabeled Amino Acids into Chlorophyll in
C. velia and Cyanobacterium Synechocystis 6803.
Cells were incubated with 350 mM 14C-Glu or 14C-Gly (specific activity
;50 mCi/mmol) for 2 h at 308C and at 80 mmol of photons s 1 m 2.
Chlorophyll was than extracted and converted into chlorin lacking both
the phytol and methyl group at isocyclic E ring; thus, contains carbons
from ALA precursors only. Prepared chlorin was separated by thin layer
chromatography and exposed to an x-ray film.
[See online article for color version of this figure.]
8 of 9
The Plant Cell
the trees were discarded). It should be noted that the presence of
Plasmodium sequences in our analyses did not support any other method
of tree construction because of their high divergence. Only the use of the
advanced CAT model supported the expected placement of Plasmodium
into the same clade with other apicomplexans in many of the analyses.
Thus, the trees show only Bayesian posterior probabilities and not any other
support, such as maximum likelihood bootstraps. We preferred to use all
apicomplexan sequences, including those that are highly divergent, before
the use of other additional phylogenetic methods on the reduced data set.
Putative Localizations of Enzymes of the Tetrapyrrole
Synthesis in C. velia
Putative localizations of nuclear encoded enzymes of the tetrapyrrole pathway were tested by in silico predictions using CBS prediction servers (http://
www.cbs.dtu.dk/services/). In particular, programs SignalP and TargetP
(Emanuelsson et al., 2007) were used to predict SPs and TPs, respectively.
In Vivo Radiolabeling of Chlorophyll
To perform in vivo radiolabeling of chlorophyll, 50 mL of C. velia or
Synechocystis 6803 cells at OD750 ;0.4 were concentrated into 350 mL of
growth media supplemented with 20 mM TES/NaOH buffer, pH 8.0,
transferred into a glass tube, and incubated on a shaker for 1 h at 308C
and at 80 mmol of photons s21 m22. Then, 350 mM 14C-Glu or 14C-Gly was
added (specific activity ;50 mCi/mmol; American Radiolabeled Chemicals), and cells were further incubated under the same conditions.
Incorporation of labeled amino acid into cells was monitored by measuring decrease of the radioactivity from the supernatant every 30 min on
liquid scintillation analyzer (Packard Tri-Carb 1500). After 2 h, cells were
washed three times with water and then pigments were extracted from
pelleted cells using 1.3 mL of methanol/0.2% NH4. This solution was
mixed with 140 mL of 1 M NaCl and 400 mL of hexane and vortexed.
Hexane phase containing chlorophyll was removed and evaporated on a
vacuum concentrator, resuspended in 100 mL of methanol, and mixed
with 100 mL of methanol containing 10% KOH. After incubation for 15 min
at room temperature, the solution was extracted twice with 200 mL
hexane and once by 200 mL of petroleum ether (boiling range 40 to 658C)
to remove phytol and unprocessed chlorophyll, and finally acidified by 20
mL of concentrated HCl. The resulting solution containing the chlorophyll
derivate chlorin was cleared by centrifugation, the supernatant transferred into a new tube, dried on a vacuum concentrator, and resuspended
in a drop of methanol. Reverse-phase thin layer chromatography was
performed on silica-gel plate (Macherey-Nagel); chloroform:methanol:
water (65:25:3) was used as the mobile phase. After separation, the plate
was dried and the signal captured on an x-ray film (exposure time of 5 d).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
data libraries under the following accession numbers: HQ222925 to
HQ222935 for the complete genes of the tetrapyrrole biosynthesis of C.
velia and HQ245653 to HQ245656 for the partial gene sequences of the
strain CCMP3155. Accession numbers for sequences used in phylogenetic analysis are presented in Supplemental Table 1 online.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Phylogenetic Trees of d-Aminolevulinic Acid
Dehydratase, Porphobilinogen Deaminase, Uroporphyrinogen Synthase,
Coproporphyrinogen Oxidase, and Protoporphyrinogen Oxidase.
Supplemental Figure 2. Comparison of ALAS N-Terminal Presequences of Various Eukaryotes, Including C. velia.
Supplemental Figure 3. Presence of Conserved Heme Regulatory
Motifs (CP Motifs) in the N-Terminal Presequences of d-Aminolevulinic Acid Synthases of Various Eukaryotes, Including C. velia.
Supplemental Table 1. Sequences Used in the Analyses.
Supplemental Data Set 1. Text File of Alignment Corresponding to
Figure 1.
Supplemental Data Set 2. Text File of Alignment Corresponding to
Figure 2.
Supplemental Data Set 3. Text File of Alignment Corresponding to
Supplemental Figure 1.
ACKNOWLEDGMENTS
This work was supported by the Czech Science Foundation (GA206/08/
1423), the Grant Agency of the Academy of Sciences of the Czech
Republic (IAA601410907), by Award IC/2010/09 made by King Abdullah
University of Science and Technology, by the project Algatech (CZ.1.05/
2.1.00/03.0110), and by the Grant Agency of University of South
Bohemia (GAJU 146/2010/P) to M.O.; by Institutional Research Concept
AV0Z50200510 to R.S.; and by the Canadian Institutes for Health
Research (MOP42517) to P.J.K. P.J.K. is a Fellow of the Canadian
Institute for Advanced Research and a Senior Scholar of the Michael
Smith Foundation for Health Research.
AUTHOR CONTRIBUTIONS
L.K., R.S., P.J.K., and M.O. designed research. L.K. and R.S. performed
research. L.K. and J.J. analyzed data. L.K. and M.O. wrote the article.
Received July 11, 2011; revised September 7, 2011; accepted September
19, 2011; published September 30, 2011.
REFERENCES
Alemu, A., Tsegaye, W., Golassa, L., and Abebe, G. (2011). Urban malaria
and associated risk factors in Jimma town, south-west Ethiopia. Malar. J.
10: 173.
Camadro, J.M., Chambon, H., Jolles, J., and Labbe, P. (1986).
Purification and properties of coproporphyrinogen oxidase from the
yeast Saccharomyces cerevisiae. Eur. J. Biochem. 156: 579–587.
Castelfranco, P.A., and Jones, O.T.G. (1975). Protoheme turnover and
chlorophyll synthesis in greening barley tissue. Plant Physiol. 55: 485–490.
Dailey, T.A., Woodruff, J.H., and Dailey, H.A. (2005). Examination of
mitochondrial protein targeting haem synthetic enzymes: in vivo
identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase. Biochem. J. 386: 381–386.
Emanuelsson, O., Brunak, S., von Heijne, G., and Nielsen, H. (2007).
Locating proteins in the cell using TargetP, SignalP and related tools.
Nat. Protoc. 2: 953–971.
Foth, B.J., Ralph, S.A., Tonkin, C.J., Struck, N.S., Fraunholz, M.,
Roos, D.S., Cowman, A.F., and McFadden, G.I. (2003). Dissecting
apicoplast targeting in the malaria parasite Plasmodium falciparum.
Science 299: 705–708.
Gould, S.B., Sommer, M.S., Hadfi, K., Zauner, S., Kroth, P.G., and
Maier, U.G. (2006). Protein targeting into the complex plastid of
cryptophytes. J. Mol. Evol. 62: 674–681.
Tetrapyrrole Synthesis in Chromera velia
Hall, T.A. (1999). BioEdit: User-friendly biological sequence alignmet
editor and analysis program for Windows 95/98/NT. Nucleic Acids Res.
41: 95–98.
Hirakawa, Y., Nagamune, K., and Ishida, K. (2009). Protein targeting
into secondary plastids of chlorarachniophytes. Proc. Natl. Acad. Sci.
USA 106: 12820–12825.
Janouškovec, J., Horák, A., Obornı́k, M., Lukeš, J., and Keeling, P.J.
(2010). A common red algal origin of the apicomplexan, dinoflagellate,
and heterokont plastids. Proc. Natl. Acad. Sci. USA 107: 10949–10954.
Jiroutová, K., Horák, A., Bowler, C., and Obornı́k, M. (2007). Tryptophan biosynthesis in stramenopiles: eukaryotic winners in the
diatom complex chloroplast. J. Mol. Evol. 65: 496–511.
Kalanon, M., and McFadden, G.I. (2010). Malaria, Plasmodium falciparum and its apicoplast. Biochem. Soc. Trans. 38: 775–782.
Katoh, K., and Toh, H. (2008). Recent developments in the MAFFT
multiple sequence alignment program. Brief. Bioinform. 9: 286–298.
Kořený, L., and Obornı́k, M. (2011). Sequence evidence for the
presence of two tetrapyrrole pathways in Euglena gracilis. Genome
Biol. Evol. 3: 359–364.
Kroth, P.G. (2002). Protein transport into secondary plastids and the evolution of primary and secondary plastids. Int. Rev. Cytol. 221: 191–255.
Lang, M., Apt, K.E., and Kroth, P.G. (1998). Protein transport into
“complex” diatom plastids utilizes two different targeting signals.
J. Biol. Chem. 273: 30973–30978.
Lartillot, N., and Philippe, H. (2004). A Bayesian mixture model for
across-site heterogeneities in the amino-acid replacement process.
Mol. Biol. Evol. 21: 1095–1109.
Mather, M.W., and Vaidya, A.B. (2008). Mitochondria in malaria and
related parasites: Ancient, diverse and streamlined. J. Bioenerg.
Biomembr. 40: 425–433.
McFadden, G.I. (1999). Plastids and protein targeting. J. Eukaryot.
Microbiol. 46: 339–346.
Mogi, T., and Kita, K. (2010). Diversity in mitochondrial metabolic
pathways in parasitic protists Plasmodium and Cryptosporidium.
Parasitol. Int. 59: 305–312.
Moore, R.B., et al. (2008). A photosynthetic alveolate closely related to
apicomplexan parasites. Nature 451: 959–963.
Nagaraj, V.A., Arumugam, R., Chandra, N.R., Prasad, D., Rangarajan,
P.N., and Padmanaban, G. (2009a). Localisation of Plasmodium
falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic
pathway in the apicoplast and characterisation of its catalytic properties.
Int. J. Parasitol. 39: 559–568.
Nagaraj, V.A., Arumugam, R., Prasad, D., Rangarajan, P.N., and
Padmanaban, G. (2010a). Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion.
Mol. Biochem. Parasitol. 174: 44–52.
Nagaraj, V.A., Prasad, D., Arumugam, R., Rangarajan, P.N., and
Padmanaban, G. (2010b). Characterization of coproporphyrinogen III
oxidase in Plasmodium falciparum cytosol. Parasitol. Int. 59: 121–127.
Nagaraj, V.A., Prasad, D., Rangarajan, P.N., and Padmanaban, G.
(2009b). Mitochondrial localization of functional ferrochelatase from
Plasmodium falciparum. Mol. Biochem. Parasitol. 168: 109–112.
Nassoury, N., Cappadocia, M., and Morse, D. (2003). Plastid ultrastructure defines the protein import pathway in dinoflagellates. J. Cell
Sci. 116: 2867–2874.
Obornı́k, M., and Green, B.R. (2005). Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes. Mol. Biol. Evol. 22: 2343–2353.
Obornı́k, M., Janouškovec, J., Chrudimský, T., and Lukeš, J. (2009).
Evolution of the apicoplast and its hosts: From heterotrophy to
autotrophy and back again. Int. J. Parasitol. 39: 1–12.
Obornı́k, M., Vancová, M., Lai, D.H., Janouškovec, J., Keeling, P.J.,
and Lukeš, J. (2011). Morphology and ultrastructure of multiple life
9 of 9
cycle stages of the photosynthetic relative of apicomplexa, Chromera
velia. Protist 162: 115–130.
Osafune, T., and Schiff, J.A. (1983). W10BSmL, a mutant of Euglena
gracilis var. bacillaris lacking plastids. Exp. Cell Res. 148: 530–535.
Panek, H., and O’Brian, M.R. (2002). A whole genome view of prokaryotic haem biosynthesis. Microbiology 148: 2273–2282.
Papenbrock, J., Mock, H.P., Kruse, E., and Grimm, B. (1999). Expression studies in tetrapyrrole biosynthesis: inverse maxima of
magnesium chelatase and ferrochelatase activity during cyclic photoperiods. Planta 208: 264–273.
Ralph, S.A., van Dooren, G.G., Waller, R.F., Crawford, M.J., Fraunholz,
M.J., Foth, B.J., Tonkin, C.J., Roos, D.S., and McFadden, G.I. (2004).
Tropical infectious diseases: Metabolic maps and functions of the
Plasmodium falciparum apicoplast. Nat. Rev. Microbiol. 2: 203–216.
Rao, A., Yeleswarapu, S.J., Srinivasan, R., and Bulusu, G. (2008).
Localization of heme biosynthesis pathway enzymes in Plasmodium
falciparum. Indian J. Biochem. Biophys. 45: 365–373.
Sato, S., Clough, B., Coates, L., and Wilson, R.J.M. (2004). Enzymes
for heme biosynthesis are found in both the mitochondrion and plastid
of the malaria parasite Plasmodium falciparum. Protist 155: 117–125.
Sato, S., and Wilson, R.J.M. (2003). Proteobacteria-like ferrochelatase
in the malaria parasite. Curr. Genet. 42: 292–300.
Shanmugam, D., Wu, B., Ramirez, U., Jaffe, E.K., and Roos, D.S.
(2010). Plastid-associated porphobilinogen synthase from Toxoplasma
gondii: Kinetic and structural properties validate therapeutic potential.
J. Biol. Chem. 285: 22122–22131.
Shashidhara, L.S., Lim, S.H., Shackleton, J.B., Robinson, C., and
Smith, A.G. (1992). Protein targeting across the three membranes of
the Euglena chloroplast envelope. J. Biol. Chem. 267: 12885–12891.
Schulze, J.O., Schubert, W.D., Moser, J., Jahn, D., and Heinz, D.W.
(2006). Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis. J. Mol. Biol. 358: 1212–1220.
Templeton, T.J., Enomoto, S., Chen, W.J., Huang, C.G., Lancto,
C.A., Abrahamsen, M.S., and Zhu, G. (2010). A genome-sequence
survey for Ascogregarina taiwanensis supports evolutionary affiliation
but metabolic diversity between a Gregarine and Cryptosporidium.
Mol. Biol. Evol. 27: 235–248.
van Dooren, G.G., Stimmler, L.M., and McFadden, G.I. (2006). Metabolic maps and functions of the Plasmodium mitochondrion. FEMS
Microbiol. Rev. 30: 596–630.
Volland, C., and Felix, F. (1984). biosynthesisIsolation and properties of
5-aminolevulinate synthase from the yeast Saccharomyces cerevisiae.
Eur. J. Biochem. 142: 551–557.
von Heijne, G., Steppuhn, J., and Herrmann, R.G. (1989). Domain
structure of mitochondrial and chloroplast targeting peptides. Eur. J.
Biochem. 180: 535–545.
Waller, R.F., Keeling, P.J., Donald, R.G.K., Striepen, B., Handman,
E., Lang-Unnasch, N., Cowman, A.F., Besra, G.S., Roos, D.S., and
McFadden, G.I. (1998). Nuclear-encoded proteins target to the
plastid in Toxoplasma gondii and Plasmodium falciparum. Proc.
Natl. Acad. Sci. USA 95: 12352–12357.
Weinstein, J.D., and Beale, S.I. (1983). Separate physiological roles
and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. J. Biol. Chem. 258: 6799–6807.
Woodson, J.D., Perez-Ruiz, J.M., and Chory, J. (2011). Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in
plants. Curr. Biol. 21: 897–903.
Wu, B. (2006). Heme Biosynthetic Pathway in Apicomplexan Parasites.
PhD dissertation (Philadelphia, PA: University of Pennsylvania).
Zhang, L., and Guarente, L. (1995). Heme binds to a short sequence
that serves a regulatory function in diverse proteins. EMBO J. 14:
313–320.
Tetrapyrrole Synthesis of Photosynthetic Chromerids Is Likely Homologous to the Unusual
Pathway of Apicomplexan Parasites
Ludek Korený, Roman Sobotka, Jan Janouskovec, Patrick J. Keeling and Miroslav Oborník
Plant Cell; originally published online September 30, 2011;
DOI 10.1105/tpc.111.089102
This information is current as of June 18, 2017
Supplemental Data
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